bims-proteo 2024-10-06 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–10–06
43 papers selected by
Eric Chevet, INSERM

TMX5/TXNDC15, a natural trapping mutant of the PDI family is a client of the proteostatic factor ERp44.
Regulation of leptin signaling and diet-induced obesity by SEL1L-HRD1 ER-associated degradation in POMC expressing neurons.
Precise Modulation of Protein Degradation by Smart PROTACs.
G3BP isoforms differentially affect stress granule assembly and gene expression during cellular stress.
Stress-Induced Eukaryotic Translational Regulatory Mechanisms.
Tapasin assembly surveillance by the RNF185/Membralin ubiquitin ligase complex regulates MHC-I surface expression.
Integrated stress response plasticity governs normal cell adaptation to chronic stress via the PP2A-TFE3-ATF4 pathway.
Ribosome Quality Control mitigates the cytotoxicity of ribosome collisions induced by 5-Fluorouracil.
Emergence and stability of endoplasmic reticulum network streaming in plant cells.
Temporal alterations of the nascent proteome in response to mitochondrial stress.
Nucleoporin Nsp1 surveils the phase state of FG-Nups.
TDRD3 functions as a selective autophagy receptor with dual roles in autophagy and modulation of stress granule stability.
Hormone-mediated disassembly and inactivation of a plant E3 ubiquitin ligase complex.
A functional screen for ubiquitin regulation identifies an E3 ligase secreted by Pseudomonas aeruginosa.
Sec23IP recruits VPS13B/COH1 to ER exit site-Golgi interface for tubular ERGIC formation.
High-grade serous ovarian cancer development and anti-PD-1 resistance is driven by IRE1α activity in neutrophils.
Evolution of the conformational dynamics of the molecular chaperone Hsp90.
Migratory autolysosome disposal mitigates lysosome damage.
CHIP ameliorates nonalcoholic fatty liver disease via promoting K63- and K27-linked STX17 ubiquitination to facilitate autophagosome-lysosome fusion.
Human DUS1L catalyzes dihydrouridine modification at tRNA positions 16/17, and DUS1L overexpression perturbs translation.
Mechanisms and regulation of substrate degradation by the 26S proteasome.
The molecular dissection of TRIM25's RNA-binding mechanism provides key insights into its antiviral activity.
Pervasive mislocalization of pathogenic coding variants underlying human disorders.
Decoding polyubiquitin regulation of K V 7. 1 functional expression with engineered linkage-selective deubiquitinases.
Purkinje Cell-specific Deficiency in SEL1L-HRD1 Endoplasmic Reticulum-Associated Degradation Causes Progressive Cerebellar Ataxia in Mice.
Plekhg5 controls the unconventional secretion of Sod1 by presynaptic secretory autophagy.
Defective regulation of the eIF2-eIF2B translational axis underlies depressive-like behavior in mice and correlates with major depressive disorder in humans.
Activation of the heat shock response as a therapeutic strategy for tau toxicity.
Understanding nuclear mRNA export: Survival under stress.
The middle domain of Hsp104 can ensure substrates are functional after processing.
Functional Diversification of Sec13 Isoforms for Storage Protein Trafficking in Rice Endosperm Cells.
Single-molecule tracking reveals dynamic regulation of ribosomal scanning.
KDELR2 is necessary for chronic obstructive pulmonary disease airway Mucin5AC hypersecretion via an IRE1α/XBP-1s-dependent mechanism.
A structural rationale for reversible vs irreversible amyloid fibril formation from a single protein.
Chemical Diversification of Enzymatically Assembled Polyubiquitin Chains to Decipher the Ubiquitin Codes Programmed on the Branch Structure.
Translational profiling of stress-induced small proteins uncovers an unexpected connection among distinct signaling systems.
Ageing-associated long non-coding RNA extends lifespan and reduces translation in non-dividing cells.
Protocol to measure and analyze protein interactions in mammalian cells using bioluminescence resonance energy transfer.
Proteome-wide CETSA reveals diverse apoptosis-inducing mechanisms converging on an initial apoptosis effector stage at the nuclear periphery.
Family-Wide Photoproximity Profiling of Integrin Protein Social Networks in Cancer.
RNA-driven phase transitions in biomolecular condensates.
Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors.
High-throughput optimized prime editing mediated endogenous protein tagging for pooled imaging of protein localization.

Life Sci Alliance. 2024 Dec;pii: e202403047. [Epub ahead of print]7(12):

TMX5/TXNDC15, a natural trapping mutant of the PDI family is a client of the proteostatic factor ERp44.

Tatiana Soldà, Carmela Galli, Concetta Guerra, Carolin Hoefner, Maurizio Molinari.

The ER is the organelle of nucleated cells that produces lipids, sugars, and proteins. More than 20 ER-resident members of the protein disulfide isomerase (PDI) family regulate formation, isomerization, and disassembly of covalent bonds in newly synthesized polypeptides. The PDI family includes few membrane-bound members. Among these, TMX1, TMX2, TMX3, TMX4, and TMX5 belong to the thioredoxin-related transmembrane (TMX) protein family. TMX5 is the least-known member of the family. Here, we establish that TMX5 covalently engages via its active site cysteine residue at position 220 a subset of secretory proteins, mainly single- and multipass Golgi-resident polypeptides. TMX5 also interacts non-covalently, and covalently, via non-catalytic cysteine residues, with the PDI family members PDI, ERp57, and ERp44. The association between TMX5 and ERp44 requires formation of a mixed disulfide between the catalytic cysteine residue 29 of ERp44 and the non-catalytic cysteine residues 114 and/or 124 of TMX5 and controls the ER localization of TMX5 in pre-Golgi compartments. Thus, TMX5 belongs to the family of proteins including Ero1α, Ero1β, Prx4, ERAP1, and SUMF1 that operate in pre-Golgi compartments but lack localization sequences required to position themselves and rely on ERp44 engagement for proper intercompartmental distribution.

DOI: https://doi.org/10.26508/lsa.202403047
Nat Commun. 2024 Sep 29. 15(1): 8435

Regulation of leptin signaling and diet-induced obesity by SEL1L-HRD1 ER-associated degradation in POMC expressing neurons.

Hancheng Mao, Geun Hyang Kim, Linxiu Pan, Ling Qi.

Endoplasmic reticulum (ER) homeostasis in the hypothalamus has been implicated in the pathogenesis of diet-induced obesity (DIO) and type 2 diabetes; however, the underlying molecular mechanism remain vague and debatable. Here we report that SEL1L-HRD1 protein complex of the highly conserved ER-associated protein degradation (ERAD) machinery in POMC-expressing neurons ameliorates diet-induced obesity and its associated complications, partly by regulating the turnover of the long isoform of Leptin receptors (LepRb). Loss of SEL1L in POMC-expressing neurons attenuates leptin signaling and predisposes mice to HFD-associated pathologies including fatty liver, glucose intolerance, insulin and leptin resistance. Mechanistically, nascent LepRb, both wildtype and disease-associated Cys604Ser variant, are misfolding prone and bona fide substrates of SEL1L-HRD1 ERAD. In the absence of SEL1L-HRD1 ERAD, LepRb are largely retained in the ER, in an ER stress-independent manner. This study uncovers an important role of SEL1L-HRD1 ERAD in the pathogenesis of central leptin resistance and leptin signaling.

DOI: https://doi.org/10.1038/s41467-024-52743-2
Chembiochem. 2024 Oct 04. e202400682

Precise Modulation of Protein Degradation by Smart PROTACs.

Junfei Cheng, Guoqiang Dong, Wei Wang, Chunquan Sheng.

  Proteolysis-targeting chimera (PROTAC) has emerged as an attractive therapeutic modality in drug discovery. PROTACs are bifunctional molecules that effectively bridge proteins of interest (POIs) with E3 ubiquitin ligases, such that, the target proteins are tagged with ubiquitin and subsequently degraded via the proteasome. Despite significant progress in the field of targeted protein degradation (TPD), the application of conventional PROTAC degraders still faces significant challenges, including systemic toxicity induced by non-tissue-specific targeting. To address this issue, a variety of smart PROTACs that can be activated by specific stimuli, have been developed for achieving conditional and spatiotemporal modulation of protein levels. Here, on the basis of our contributions, we overview recent advances of smart PROTACs, including tumor microenvironment-, photo-, and X-ray radiation-responsive PROTACs, that enable controllable TPD. The design strategy, case studies, potential applications and challenges will be focused on.

Keywords:  drug discovery; off-tissue toxicity; precision cancer therapy; smart PROTACs; targeted protein degradation

DOI:  https://doi.org/10.1002/cbic.202400682
Mol Biol Cell. 2024 Oct 02. mbcE24020062

G3BP isoforms differentially affect stress granule assembly and gene expression during cellular stress.

José M Liboy-Lugo, Carla A Espinoza, Jessica Sheu-Gruttadauria, Jesslyn E Park, Albert Xu, Ziad Jowhar, Angela L Gao, José A Carmona-Negrón, Torsten Wittmann, Natalia Jura, Stephen N Floor.

Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these membrane-less organelles is driven by the condensation of RNA and RNA-binding proteins such as G3BPs. G3BPs form SGs following stress-induced translational arrest. Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. However, the contribution of different G3BP paralogs to stress granule formation and gene expression changes is incompletely understood. Here, we probed the functions of G3BPs by identifying important residues for stress granule assembly at their N-terminal domain such as V11. This conserved amino acid is required for formation of the G3BP-Caprin-1 complex, hence promoting SG assembly. Total RNA sequencing and ribosome profiling revealed that a G3BPV11A mutant leads to changes in mRNA levels and ribosome engagement during the integrated stress response (ISR). Moreover, we found that G3BP2B preferentially forms stress granules and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. Furthermore, our work is a resource for researchers to study gene expression changes under cellular stress. Together, this work suggests that perturbing protein-protein interactions mediated by G3BPs affect stress granule assembly and gene expression during the ISR, and such functions are differentially regulated by G3BP paralogs under ER stress.

DOI: https://doi.org/10.1091/mbc.E24-02-0062
J Clin Med Sci. 2024 ;pii: 1000277. [Epub ahead of print]8(2):

Stress-Induced Eukaryotic Translational Regulatory Mechanisms.

Dilawar Ahmad Mir, Zhengxin Ma, Jordan Horrocks, Aric Rogers.

The eukaryotic protein synthesis process entails intricate stages governed by diverse mechanisms to tightly regulate translation. Translational regulation during stress is pivotal for maintaining cellular homeostasis, ensuring the accurate expression of essential proteins is important for survival. This selective translational control mechanism is integral to cellular adaptation and resilience under adverse conditions. This review manuscript explores various mechanisms involved in selective translational regulation, focusing on mRNA-specific and global regulatory processes. Key aspects of translational control include translation initiation, which is often a rate-limiting step, and involves the formation of the eIF4F complex and recruitment of mRNA to ribosomes. Regulation of translation initiation factors, such as eIF4E, eIF4E2, and eIF2, through phosphorylation and interactions with binding proteins, modulates translation efficiency under stress conditions. This review also highlights the control of translation initiation through factors like the eIF4F complex and the ternary complex and also underscores the importance of eIF2α phosphorylation in stress granule formation and cellular stress responses. Additionally, the impact of amino acid deprivation, mTOR signaling, and ribosome biogenesis on translation regulation and cellular adaptation to stress is also discussed. Understanding the intricate mechanisms of translational regulation during stress provides insights into cellular adaptation mechanisms and potential therapeutic targets for various diseases, offering valuable avenues for addressing conditions associated with dysregulated protein synthesis.

Keywords: Signaling; Stress; Translation regulations mechanisms; mRNA; mTOR
Nat Commun. 2024 Oct 01. 15(1): 8508

Tapasin assembly surveillance by the RNF185/Membralin ubiquitin ligase complex regulates MHC-I surface expression.

Michael L van de Weijer, Krishna Samanta, Nikita Sergejevs, LuLin Jiang, Maria Emilia Dueñas, Tiaan Heunis, Timothy Y Huang, Randal J Kaufman, Matthias Trost, Sumana Sanyal, Sally A Cowley, Pedro Carvalho.

Immune surveillance by cytotoxic T cells eliminates tumor cells and cells infected by intracellular pathogens. This process relies on the presentation of antigenic peptides by Major Histocompatibility Complex class I (MHC-I) at the cell surface. The loading of these peptides onto MHC-I depends on the peptide loading complex (PLC) at the endoplasmic reticulum (ER). Here, we uncovered that MHC-I antigen presentation is regulated by ER-associated degradation (ERAD), a protein quality control process essential to clear misfolded and unassembled proteins. An unbiased proteomics screen identified the PLC component Tapasin, essential for peptide loading onto MHC-I, as a substrate of the RNF185/Membralin ERAD complex. Loss of RNF185/Membralin resulted in elevated Tapasin steady state levels and increased MHC-I at the surface of professional antigen presenting cells. We further show that RNF185/Membralin ERAD complex recognizes unassembled Tapasin and limits its incorporation into PLC. These findings establish a novel mechanism controlling antigen presentation and suggest RNF185/Membralin as a potential therapeutic target to modulate immune surveillance.

DOI: https://doi.org/10.1038/s41467-024-52772-x
Cell Death Differ. 2024 Sep 30.

Integrated stress response plasticity governs normal cell adaptation to chronic stress via the PP2A-TFE3-ATF4 pathway.

Rita A Avelar, Riya Gupta, Grace Carvette, Felipe da Veiga Leprevost, Medhasri Jasti, Jose Colina, Jessica Teitel, Alexey I Nesvizhskii, Caitlin M O'Connor, Maria Hatzoglou, Shirish Shenolikar, Peter Arvan, Goutham Narla, Analisa DiFeo.

The integrated stress response (ISR) regulates cell fate during conditions of stress by leveraging the cell's capacity to endure sustainable and efficient adaptive stress responses. Protein phosphatase 2A (PP2A) activity modulation has been shown to be successful in achieving both therapeutic efficacy and safety across various cancer models. However, the molecular mechanisms driving its selective antitumor effects remain unclear. Here, we show for the first time that ISR plasticity relies on PP2A activation to regulate drug response and dictate cellular survival under conditions of chronic stress. We demonstrate that genetic and chemical modulation of the PP2A leads to chronic proteolytic stress and triggers an ISR to dictate whether the cell lives or dies. More specifically, we uncovered that the PP2A-TFE3-ATF4 pathway governs ISR cell plasticity during endoplasmic reticular and cellular stress independent of the unfolded protein response. We further show that normal cells reprogram their genetic signatures to undergo ISR-mediated adaptation and homeostatic recovery thereby avoiding toxicity following PP2A-mediated stress. Conversely, oncogenic specific cytotoxicity induced by chemical modulation of PP2A is achieved by activating chronic and irreversible ISR in cancer cells. Our findings propose that a differential response to chemical modulation of PP2A is determined by intrinsic ISR plasticity, providing a novel biological vulnerability to selectively induce cancer cell death and improve targeted therapeutic efficacy.

DOI: https://doi.org/10.1038/s41418-024-01378-3
Nucleic Acids Res. 2024 Oct 01. pii: gkae849. [Epub ahead of print]

Ribosome Quality Control mitigates the cytotoxicity of ribosome collisions induced by 5-Fluorouracil.

Susanta Chatterjee, Parisa Naeli, Okan Onar, Nicole Simms, Aitor Garzia, Angela Hackett, Kelsey Coyle, Patric Harris Snell, Tom McGirr, Tanvi Nitin Sawant, Kexin Dang, Zornitsa Vasileva Stoichkova, Yumna Azam, Mark P Saunders, Michael Braun, Tommy Alain, Thomas Tuschl, Simon S McDade, Daniel B Longley, Christos G Gkogkas, Colin Adrain, John R P Knight, Seyed Mehdi Jafarnejad.

Ribosome quality control (RQC) resolves collided ribosomes, thus preventing their cytotoxic effects. The chemotherapeutic agent 5-Fluorouracil (5FU) is best known for its misincorporation into DNA and inhibition of thymidylate synthase. However, while a major determinant of 5FU's anticancer activity is its misincorporation into RNAs, the mechanisms by which cancer cells overcome the RNA-dependent 5FU toxicity remain ill-defined. Here, we report a role for RQC in mitigating the cytotoxic effects of 5FU. We show that 5FU treatment results in rapid induction of the mTOR signalling pathway, enhanced rate of mRNA translation initiation, and increased ribosome collisions. Consistently, a defective RQC exacerbates the 5FU-induced cell death, which is mitigated by blocking mTOR pathway or mRNA translation initiation. Furthermore, 5FU treatment enhances the expression of the key RQC factors ZNF598 and GIGYF2 via an mTOR-dependent post-translational mechanism. This adaptation likely mitigates the cytotoxic consequences of increased ribosome collisions upon 5FU treatment.

DOI: https://doi.org/10.1093/nar/gkae849
J Theor Biol. 2024 Sep 27. pii: S0022-5193(24)00239-X. [Epub ahead of print] 111954

Emergence and stability of endoplasmic reticulum network streaming in plant cells.

Graham M Donovan, Congping Lin, Imogen Sparkes, Peter Ashwin.

  The endoplasmic reticulum (ER) network is highly complex and highly dynamic in its geometry, and undergoes extensive remodeling and bulk flow. It is known that the ER dynamics are driven by actin-myosin dependent processes. ER motion through the cytoplasm will cause forces on the cytoplasm that will induce flow. However, ER will also clearly be passively transported by the bulk cytoplasmic streaming. We take the complex ER network structure into account and propose a positive-feedback mechanism among myosin-like motors, actin alignment, ER network dynamics for the emergence of ER flow. Using this model, we demonstrate that ER streaming may be an emergent feature of this three-way interaction and that the persistent-point density may be a key driver of the emergence of ER streaming.

Keywords:  Actin; Cytoplasmic streaming; ER streaming; Molecular motor

DOI:  https://doi.org/10.1016/j.jtbi.2024.111954
Cell Rep. 2024 Oct 02. pii: S2211-1247(24)01154-9. [Epub ahead of print]43(10): 114803

Temporal alterations of the nascent proteome in response to mitochondrial stress.

Tomasz M Stępkowski, Vanessa Linke, Dorota Stadnik, Maciej Zakrzewski, Anna E Zawada, Remigiusz A Serwa, Agnieszka Chacinska.

  Under stress, protein synthesis is attenuated to preserve energy and mitigate challenges to protein homeostasis. Here, we describe, with high temporal resolution, the dynamic landscape of changes in the abundance of proteins synthesized upon stress from transient mitochondrial inner membrane depolarization. This nascent proteome was altered when global translation was attenuated by stress and began to normalize as translation was recovering. This transition was associated with a transient desynchronization of cytosolic and mitochondrial translation and recovery of cytosolic and mitochondrial ribosomal proteins. Further, the elongation factor EEF1A1 was downregulated upon mitochondrial stress, and its silencing mimicked the stress-induced nascent proteome remodeling, including alterations in the nascent respiratory chain proteins. Unexpectedly, the stress-induced alterations in the nascent proteome were independent of physiological protein abundance and turnover. In summary, we provide insights into the physiological and pathological consequences of mitochondrial function and dysfunction.

Keywords:  CP: Cell biology; CP: Metabolism; EEF1A; EEF1A1; cellular stress; elongation factor; mass spectrometry; mitochondria; nascent chain; protein synthesis; proteomics; translation

DOI:  https://doi.org/10.1016/j.celrep.2024.114803
Cell Rep. 2024 Oct 01. pii: S2211-1247(24)01144-6. [Epub ahead of print]43(10): 114793

Nucleoporin Nsp1 surveils the phase state of FG-Nups.

Tegan A Otto, Tessa Bergsma, Maurice Dekker, Sara N Mouton, Paola Gallardo, Justina C Wolters, Anton Steen, Patrick R Onck, Liesbeth M Veenhoff.

  Transport through the nuclear pore complex (NPC) relies on intrinsically disordered FG-nucleoporins (FG-Nups) forming a selective barrier. Away from the NPC, FG-Nups readily form condensates and aggregates, and we address how this behavior is surveilled in cells. FG-Nups, including Nsp1, together with the nuclear transport receptor Kap95, form a native daughter cell-specific cytosolic condensate in yeast. In aged cells, this condensate disappears as cytosolic Nsp1 levels decline. Biochemical assays and modeling show that Nsp1 is a modulator of FG-Nup condensates, promoting a liquid-like state. Nsp1's presence in the cytosol and condensates is critical, as a reduction of cytosolic levels in young cells induces NPC defects and a general decline in protein quality control that quantitatively mimics aging phenotypes. These phenotypes can be rescued by a cytosolic form of Nsp1. We conclude that Nsp1 is a phase state regulator that surveils FG-Nups and impacts general protein homeostasis.

Keywords:  CP: Molecular biology; FG-nucleoporin; Kap95; aging; chaperones; condensate; intrinsically disordered protein; liquid-liquid phase separation; nsp1; nuclear pore complex; protein homeostasis

DOI:  https://doi.org/10.1016/j.celrep.2024.114793
bioRxiv. 2024 Sep 22. pii: 2024.09.22.614367. [Epub ahead of print]

TDRD3 functions as a selective autophagy receptor with dual roles in autophagy and modulation of stress granule stability.

Matthew Deater, Richard E Lloyd.

Tudor Domain Containing 3 (TDRD3) is a methylarginine-reader protein that functions as a scaffold in the nucleus facilitating transcription, however TDRD3 is also recruited to stress granules (SGs) during the Integrated Stress Response (ISR) although its function therein remains largely unknown. We previously showed that TDRD3 is a novel antiviral restriction factor that is cleaved by virus 2A protease, and plays complex modulatory roles in both interferon and inflammatory signaling during stress and enterovirus infections. Here we have found that TDRD3 contains structural motifs similar to known selective autophagy receptors such as p62/SQSTM1, sharing ubiquitin associated domains (UBA) and LC3 interacting regions (LIR) that anchor cargo destined for autophagosomes to activated LC3 protein coating autophagosome membranes. This is of interest since enteroviruses hijack autophagy machinery to facilitate formation of viral replication factories, virus assembly and egress from the infected cell. Here we explored possible roles of TDRD3 in autophagy, hypothesizing that TDRD3 may function as a specialized selective autophagy receptor. We found that KO of TDRD3 in HeLa cells significantly reduces starvation induced autophagy, while its reintroduction restores it in a dose-dependent manner. Autophagy receptors are degraded during autophagy and expression levels decrease during this time. We found that TDRD3 levels decrease to the same extent as the autophagy receptor p62/SQSTM1 during autophagy, indicating autophagy-targeted turnover in that role. Knockout of TDRD3 or G3BP1 did not make significant changes in overall cell localization of LC3B or p62/SQSTM1, but did result in greater concentration of Lamp2 phagosome marker for phagosomes and phagolysosomes. To test the potential roles of TDRD3 in autophagic processes, we created a series of deletion mutants of TDRD3 lacking either UBA domain or the various LIR motifs that are predicted to interact with LC3B. Microscopic examination of starved cells expressing these variants of TDRD3 showed ΔLIR-TDRD3 had defects in colocalization with LC3B or Lamp2. Further, super resolution microscopy revealed ring structures with TDRD3 interfacing with p62/SQSTM1. In examination of arsenite induced stress granules we found recruitment of TDRD3 variants disrupted normally tight SG condensation, altered the decay rate of SGs upon release from stress and the kinetics of SG formation. We found evidence that the LIR3 motif on TDRD3 is involved in TDRD3 interaction with LC3B in coIP experiments, colocalization studies, and that this motif plays a key role in TDRD3 recruitment to SGs and SG resolution. Overall, these data support a functional role of TDRD3 in selective autophagy in a mode similar to p62/SQSTM1, with specific roles in SG stability and turnover. Enterovirus cleavage of TDRD3 likely affects both antiviral and autophagic responses that the virus controls for replication.

DOI: https://doi.org/10.1101/2024.09.22.614367
Cell Rep. 2024 Oct 03. pii: S2211-1247(24)01153-7. [Epub ahead of print]43(10): 114802

Hormone-mediated disassembly and inactivation of a plant E3 ubiquitin ligase complex.

Cristina Martínez, Elisa Iniesto, Marta García-León, Daniel García-Corredera, Sandra Fonseca, César Santiago, Mei Yang, Renbo Yu, Haodong Chen, Eva Altmann, Martin Renatus, Xing Wang Deng, Vicente Rubio.

  Phytohormone abscisic acid (ABA) regulates key plant development and environmental stress responses. The ubiquitin-proteasome system tightly controls ABA signaling. CULLIN4-RING (CRL4) E3 ubiquitin ligases use the substrate receptor module CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP10)-DDB1-DET1-DDA1 (CDDD) to target Arabidopsis ABA receptor PYL8, acting as negative regulators of ABA responses. Conversely, ABA treatment attenuates PYL8 receptor degradation, although the molecular mechanism remained elusive. Here, we show that ABA promotes the disruption of CRL4-CDDD complexes, leading to PYL8 stabilization. ABA-mediated CRL4-CDDD dissociation likely involves an altered association between DDA1-containing complexes and the COP9 signalosome (CSN), a master regulator of the assembly of cullin-based E3 ligases, including CRL4-CDDD. Indeed, treatment with CSN inhibitor CSN5i-3 suppresses the ABA effect on CRL4-CDDD assembly. Our findings indicate that ABA stabilizes PYL8 by altering the dynamics of the CRL4-CDDD-CSN complex association, showing a regulatory mechanism by which a plant hormone inhibits an E3 ubiquitin ligase to protect its own receptors from degradation.

Keywords:  ABA receptor; ABA signaling; Arabidopsis; CP: Molecular biology; CP: Plants; CRL4 E3 ligases; CSN; CSN5 inhibitor; DDA1; PYL8; PYR/PYL/RCAR; plant proteostasis

DOI:  https://doi.org/10.1016/j.celrep.2024.114802
bioRxiv. 2024 Sep 18. pii: 2024.09.18.613774. [Epub ahead of print]

A functional screen for ubiquitin regulation identifies an E3 ligase secreted by Pseudomonas aeruginosa.

Cameron G Roberts, Supender Kaur, Aaron J Ogden, Michael E Divine, Gus D Warren, Donghoon Kang, Natalia V Kirienko, Paul P Geurink, Monique P C Mulder, Ernesto S Nakayasu, Jason E McDermott, Joshua N Adkins, Alejandro Aballay, Jonathan N Pruneda.

Ubiquitin signaling controls many aspects of eukaryotic biology, including targeted protein degradation and immune defense. Remarkably, invading bacterial pathogens have adapted secreted effector proteins that hijack host ubiquitination to gain control over host responses. These ubiquitin-targeted effectors can exhibit, for example, E3 ligase or deubiquitinase activities, often without any sequence or structural homology to eukaryotic ubiquitin regulators. Such convergence in function poses a challenge to the discovery of additional bacterial virulence factors that target ubiquitin. To overcome this, we have developed a workflow to harvest natively secreted bacterial effectors and functionally screen them for ubiquitin regulatory activities. After benchmarking this approach on diverse ligase and deubiquitinase activities from Salmonella Typhimurium, Enteropathogenic Escherichia coli , and Shigella flexneri , we applied it to the identification of a cryptic E3 ligase activity secreted by Pseudomonas aeruginosa . We identified an unreported P. aeruginosa E3 ligase, which we have termed Pseudomonas Ub ligase 1 (PUL-1), that resembles none of the other E3 ligases previously established in or outside of the eukaryotic system. Importantly, in an animal model of P. aeruginosa infection, PUL-1 ligase activity plays an important role in regulating virulence. Thus, our workflow for the functional identification of ubiquitin-targeted effector proteins carries promise for expanding our appreciation of how host ubiquitin regulation contributes to bacterial pathogenesis.

DOI: https://doi.org/10.1101/2024.09.18.613774
J Cell Biol. 2024 Dec 02. pii: e202402083. [Epub ahead of print]223(12):

Sec23IP recruits VPS13B/COH1 to ER exit site-Golgi interface for tubular ERGIC formation.

Yuanjiao Du, Xinyu Fan, Chunyu Song, Weiping Chang, Juan Xiong, Lin Deng, Wei-Ke Ji.

VPS13B/COH1 is the only known causative factor for Cohen syndrome, an early-onset autosomal recessive developmental disorder with intellectual inability, developmental delay, joint hypermobility, myopia, and facial dysmorphism as common features, but the molecular basis of VPS13B/COH1 in pathogenesis remains largely unclear. Here, we identify Sec23 interacting protein (Sec23IP) at the ER exit site (ERES) as a VPS13B adaptor that recruits VPS13B to ERES-Golgi interfaces. VPS13B interacts directly with Sec23IP via the VPS13 adaptor binding domain (VAB), and the interaction promotes the association between ERES and the Golgi. Disease-associated missense mutations of VPS13B-VAB impair the interaction with Sec23IP. Knockout of VPS13B or Sec23IP blocks the formation of tubular ERGIC, an unconventional cargo carrier that expedites ER-to-Golgi transport. In addition, depletion of VPS13B or Sec23IP delays ER export of procollagen, suggesting a link between procollagen secretion and joint laxity in patients with Cohen disease. Together, our study reveals a crucial role of VPS13B-Sec23IP interaction at the ERES-Golgi interface in the pathogenesis of Cohen syndrome.

DOI: https://doi.org/10.1083/jcb.202402083
Oncoimmunology. 2024 ;13(1): 2411070

High-grade serous ovarian cancer development and anti-PD-1 resistance is driven by IRE1α activity in neutrophils.

Alexander Emmanuelli, Camilla Salvagno, Sung-Min Hwang, Deepika Awasthi, Tito A Sandoval, Chang-Suk Chae, Jin-Gyu Cheong, Chen Tan, Takao Iwawaki, Juan R Cubillos-Ruiz.

  High-grade serious ovarian cancer (HGSOC) is an aggressive malignancy that remains refractory to current immunotherapies. While advanced stage disease has been extensively studied, the cellular and molecular mechanisms that promote early immune escape in HGSOC remain largely unexplored. Here, we report that primary HGSO tumors program neutrophils to inhibit T cell anti-tumor function by activating the endoplasmic reticulum (ER) stress sensor IRE1α. We found that intratumoral neutrophils exhibited overactivation of ER stress response markers compared with their counterparts at non-tumor sites. Selective deletion of IRE1α in neutrophils delayed primary ovarian tumor growth and extended the survival of mice with HGSOC by enabling early T cell-mediated tumor control. Notably, loss of IRE1α in neutrophils sensitized tumor-bearing mice to PD-1 blockade, inducing HGSOC regression and long-term survival in ~ 50% of the treated hosts. Hence, neutrophil-intrinsic IRE1α facilitates early adaptive immune escape in HGSOC and targeting this ER stress sensor might be used to unleash endogenous and immunotherapy-elicited immunity that controls metastatic disease.

Keywords:  ER stress; IRE1; PD-1 blockade; immunotherapy; neutrophils; ovarian cancer

DOI:  https://doi.org/10.1080/2162402X.2024.2411070
Nat Commun. 2024 Oct 04. 15(1): 8627

Evolution of the conformational dynamics of the molecular chaperone Hsp90.

Stefan Riedl, Ecenaz Bilgen, Ganesh Agam, Viivi Hirvonen, Alexander Jussupow, Franziska Tippl, Maximilian Riedl, Andreas Maier, Christian F W Becker, Ville R I Kaila, Don C Lamb, Johannes Buchner.

Hsp90 is a molecular chaperone of central importance for protein homeostasis in the cytosol of eukaryotic cells, with key functional and structural traits conserved from yeast to man. During evolution, Hsp90 has gained additional functional importance, leading to an increased number of interacting co-chaperones and client proteins. Here, we show that the overall conformational transitions coupled to the ATPase cycle of Hsp90 are conserved from yeast to humans, but cycle timing as well as the dynamics are significantly altered. In contrast to yeast Hsp90, the human Hsp90 is characterized by broad ensembles of conformational states, irrespective of the absence or presence of ATP. The differences in the ATPase rate and conformational transitions between yeast and human Hsp90 are based on two residues in otherwise conserved structural elements that are involved in triggering structural changes in response to ATP binding. The exchange of these two mutations allows swapping of the ATPase rate and of the conformational transitions between human and yeast Hsp90. Our combined results show that Hsp90 evolved to a protein with increased conformational dynamics that populates ensembles of different states with strong preferences for the N-terminally open, client-accepting states.

DOI: https://doi.org/10.1038/s41467-024-52995-y
J Cell Biol. 2024 Dec 02. pii: e202403195. [Epub ahead of print]223(12):

Migratory autolysosome disposal mitigates lysosome damage.

Takami Sho, Ying Li, Haifeng Jiao, Li Yu.

Lysosomes, essential for intracellular degradation and recycling, employ damage-control strategies such as lysophagy and membrane repair mechanisms to maintain functionality and cellular homeostasis. Our study unveils migratory autolysosome disposal (MAD), a response to lysosomal damage where cells expel LAMP1-LC3 positive structures via autolysosome exocytosis, requiring autophagy machinery, SNARE proteins, and cell migration. This mechanism, crucial for mitigating lysosomal damage, underscores the role of cell migration in lysosome damage control and facilitates the release of small extracellular vesicles, highlighting the intricate relationship between cell migration, organelle quality control, and extracellular vesicle release.

DOI: https://doi.org/10.1083/jcb.202403195
Nat Commun. 2024 Oct 02. 15(1): 8519

CHIP ameliorates nonalcoholic fatty liver disease via promoting K63- and K27-linked STX17 ubiquitination to facilitate autophagosome-lysosome fusion.

Hyunjin Rho, Seungyeon Kim, Seung Up Kim, Jeong Won Kim, Sang Hoon Lee, Sang Hoon Park, Freddy E Escorcia, Joon-Yong Chung, Jaewhan Song.

The fusion of autophagosomes and lysosomes is essential for the prevention of nonalcoholic fatty liver disease (NAFLD). Here, we generate a hepatocyte-specific CHIP knockout (H-KO) mouse model that develops NAFLD more rapidly in response to a high-fat diet (HFD) or high-fat, high-fructose diet (HFHFD). The accumulation of P62 and LC3 in the livers of H-KO mice and CHIP-depleted cells indicates the inhibition of autophagosome-lysosome fusion. AAV8-mediated overexpression of CHIP in the murine liver slows the progression of NAFLD induced by HFD or HFHFD feeding. Mechanistically, CHIP induced K63- and K27-linked polyubiquitination at the lysine 198 residue of STX17, resulting in increased STX17-SNAP29-VAMP8 complex formation. The STX17 K198R mutant was not ubiquitinated by CHIP; it interfered with its interaction with VAMP8, rendering STX17 incapable of inhibiting steatosis development in mice. These results indicate that a signaling regulatory mechanism involving CHIP-mediated non-degradative ubiquitination of STX17 is necessary for autophagosome-lysosome fusion.

DOI: https://doi.org/10.1038/s41467-024-53002-0
Commun Biol. 2024 Oct 02. 7(1): 1238

Human DUS1L catalyzes dihydrouridine modification at tRNA positions 16/17, and DUS1L overexpression perturbs translation.

Jin Matsuura, Shinichiro Akichika, Fan-Yan Wei, Tsutomu Suzuki, Takahiro Yamamoto, Yuka Watanabe, Leoš Shivaya Valášek, Akitake Mukasa, Kazuhito Tomizawa, Takeshi Chujo.

Human cytoplasmic tRNAs contain dihydrouridine modifications at positions 16 and 17 (D16/D17). The enzyme responsible for D16/D17 formation and its cellular roles remain elusive. Here, we identify DUS1L as the human tRNA D16/D17 writer. DUS1L knockout in the glioblastoma cell lines LNZ308 and U87 causes loss of D16/D17. D formation is reconstituted in vitro using recombinant DUS1L in the presence of NADPH or NADH. DUS1L knockout/overexpression in LNZ308 cells shows that DUS1L supports cell growth. Moreover, higher DUS1L expression in glioma patients is associated with poorer prognosis. Upon vector-mediated DUS1L overexpression in LNZ308 cells, 5' and 3' processing of precursor tRNATyr(GUA) is inhibited, resulting in a reduced mature tRNATyr(GUA) level, reduced translation of the tyrosine codons UAC and UAU, and reduced translational readthrough of the near-cognate stop codons UAA and UAG. Moreover, DUS1L overexpression increases the amounts of several D16/D17-containing tRNAs and total cellular translation. Our study identifies a human dihydrouridine writer, providing the foundation to study its roles in health and disease.

DOI: https://doi.org/10.1038/s42003-024-06942-8
Nat Rev Mol Cell Biol. 2024 Oct 03.

Mechanisms and regulation of substrate degradation by the 26S proteasome.

Connor Arkinson, Ken C Dong, Christine L Gee, Andreas Martin.

The 26S proteasome is involved in degrading and regulating the majority of proteins in eukaryotic cells, which requires a sophisticated balance of specificity and promiscuity. In this Review, we discuss the principles that underly substrate recognition and ATP-dependent degradation by the proteasome. We focus on recent insights into the mechanisms of conventional ubiquitin-dependent and ubiquitin-independent protein turnover, and discuss the plethora of modulators for proteasome function, including substrate-delivering cofactors, ubiquitin ligases and deubiquitinases that enable the targeting of a highly diverse substrate pool. Furthermore, we summarize recent progress in our understanding of substrate processing upstream of the 26S proteasome by the p97 protein unfoldase. The advances in our knowledge of proteasome structure, function and regulation also inform new strategies for specific inhibition or harnessing the degradation capabilities of the proteasome for the treatment of human diseases, for instance, by using proteolysis targeting chimera molecules or molecular glues.

DOI: https://doi.org/10.1038/s41580-024-00778-0
Nat Commun. 2024 Oct 01. 15(1): 8485

The molecular dissection of TRIM25's RNA-binding mechanism provides key insights into its antiviral activity.

Lucía Álvarez, Kevin Haubrich, Louisa Iselin, Laurent Gillioz, Vincenzo Ruscica, Karine Lapouge, Sandra Augsten, Ina Huppertz, Nila Roy Choudhury, Bernd Simon, Pawel Masiewicz, Mathilde Lethier, Stephen Cusack, Katrin Rittinger, Frank Gabel, Alexander Leitner, Gracjan Michlewski, Matthias W Hentze, Frédéric H T Allain, Alfredo Castello, Janosch Hennig.

TRIM25 is an RNA-binding ubiquitin E3 ligase with central but poorly understood roles in the innate immune response to RNA viruses. The link between TRIM25's RNA binding and its role in innate immunity has not been established. Thus, we utilized a multitude of biophysical techniques to identify key RNA-binding residues of TRIM25 and developed an RNA-binding deficient mutant (TRIM25-m9). Using iCLIP2 in virus-infected and uninfected cells, we identified TRIM25's RNA sequence and structure specificity, that it binds specifically to viral RNA, and that the interaction with RNA is critical for its antiviral activity.

DOI: https://doi.org/10.1038/s41467-024-52918-x
Cell. 2024 Sep 25. pii: S0092-8674(24)01021-3. [Epub ahead of print]

Pervasive mislocalization of pathogenic coding variants underlying human disorders.

Jessica Lacoste, Marzieh Haghighi, Shahan Haider, Chloe Reno, Zhen-Yuan Lin, Dmitri Segal, Wesley Wei Qian, Xueting Xiong, Tanisha Teelucksingh, Esteban Miglietta, Hamdah Shafqat-Abbasi, Pearl V Ryder, Rebecca Senft, Beth A Cimini, Ryan R Murray, Chantal Nyirakanani, Tong Hao, Gregory G McClain, Frederick P Roth, Michael A Calderwood, David E Hill, Marc Vidal, S Stephen Yi, Nidhi Sahni, Jian Peng, Anne-Claude Gingras, Shantanu Singh, Anne E Carpenter, Mikko Taipale.

Widespread sequencing has yielded thousands of missense variants predicted or confirmed as disease causing. This creates a new bottleneck: determining the functional impact of each variant-typically a painstaking, customized process undertaken one or a few genes and variants at a time. Here, we established a high-throughput imaging platform to assay the impact of coding variation on protein localization, evaluating 3,448 missense variants of over 1,000 genes and phenotypes. We discovered that mislocalization is a common consequence of coding variation, affecting about one-sixth of all pathogenic missense variants, all cellular compartments, and recessive and dominant disorders alike. Mislocalization is primarily driven by effects on protein stability and membrane insertion rather than disruptions of trafficking signals or specific interactions. Furthermore, mislocalization patterns help explain pleiotropy and disease severity and provide insights on variants of uncertain significance. Our publicly available resource extends our understanding of coding variation in human diseases.

DOI: https://doi.org/10.1016/j.cell.2024.09.003
bioRxiv. 2024 Sep 17. pii: 2024.09.17.613539. [Epub ahead of print]

Decoding polyubiquitin regulation of K V 7. 1 functional expression with engineered linkage-selective deubiquitinases.

Sri Karthika Shanmugam, Scott A Kanner, Xinle Zou, Enoch Amarh, Papiya Choudhury, Rajesh Soni, Robert S Kass, Henry M Colecraft.

Protein posttranslational modification with distinct polyubiquitin linkage chains is a critical component of the 'ubiquitin code' that universally regulates protein expression and function to control biology. Functional consequences of diverse polyubiquitin linkages on proteins are mostly unknown, with progress hindered by a lack of methods to specifically tune polyubiquitin linkages on individual proteins in live cells. Here, we bridge this gap by exploiting deubiquitinases (DUBs) with preferences for hydrolyzing different polyubiquitin linkages: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K29/K33; and USP21 - non-specific. We developed a suite of engineered deubiquitinases (enDUBs) comprised of DUB catalytic domains fused to a GFP- targeted nanobody and used them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. Mass spectrometry of YFP-KCNQ1 expressed in HEK293 cells indicated channel polyubiquitination with K48 (72%) and K63 (24%) linkages being dominant. NEDD4-2 and ITCH both decreased KCNQ1 functional expression but with distinctive polyubiquitination signatures. All enDUBs reduced KCNQ1 ubiquitination but yielded unique effects on channel expression, surface density, ionic currents, and subcellular localization. The pattern of outcomes indicates K11, K29/K33, and K63 chains mediate net KCNQ1-YFP intracellular retention, but achieved in different ways: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling. The pattern of enDUB effects on KCNQ1-YFP differed in cardiomyocytes, emphasizing ubiquitin code mutability. Surprisingly, enDUB-O4 decreased KCNQ1-YFP surface density suggesting a role for K48 in forward trafficking. Lastly, linkage-selective enDUBs displayed varying capabilities to rescue distinct trafficking-deficient long QT syndrome type 1 mutations. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 functional expression, demonstrate ubiquitin code plasticity, and introduce linkage-selective enDUBs as a potent tool to help demystify the polyubiquitin code.

DOI: https://doi.org/10.1101/2024.09.17.613539
JCI Insight. 2024 Oct 01. pii: e174725. [Epub ahead of print]

Purkinje Cell-specific Deficiency in SEL1L-HRD1 Endoplasmic Reticulum-Associated Degradation Causes Progressive Cerebellar Ataxia in Mice.

Mauricio Torres, Brent Pederson, Hui Wang, Liangguang L Lin, Huilun Wang, Amara Bugarin-Lapuz, Zhen Zhao, Ling Qi.

  Recent studies have identified multiple genetic variants of SEL1L-HRD1 ER-associated degradation (ERAD) in humans with neurodevelopmental disorders and locomotor dysfunctions, including ataxia. However, the relevance and importance of SEL1L-HRD1 ERAD in the pathogenesis of ataxia remain unexplored. Here we show that SEL1L deficiency in Purkinje cells leads to early-onset progressive cerebellar ataxia with progressive loss of Purkinje cells with age. Mice with Purkinje cell-specific deletion of SEL1L (Sel1LPcp2Cre) exhibit motor dysfunction beginning around 9 weeks of age. Transmission electron microscopy (TEM) analysis reveals dilated ER and fragmented nuclei in Purkinje cells of adult Sel1LPcp2Cre mice, indicative of altered ER homeostasis and cell death. Lastly, loss of Purkinje cells is associated with a secondary neurodegeneration of granular cells, as well as robust activation of astrocytes and proliferation of microglia, in the cerebellum of Sel1LPcp2Cre mice. These data demonstrate the pathophysiological importance of SEL1L-HRD1 ERAD in Purkinje cells in the pathogenesis of cerebellar ataxia.

Keywords:  Neurodegeneration; Neuroscience

DOI:  https://doi.org/10.1172/jci.insight.174725
Nat Commun. 2024 Oct 04. 15(1): 8622

Plekhg5 controls the unconventional secretion of Sod1 by presynaptic secretory autophagy.

Amy-Jayne Hutchings, Bita Hambrecht, Alexander Veh, Neha Jadhav Giridhar, Abdolhossein Zare, Christina Angerer, Thorben Ohnesorge, Maren Schenke, Bhuvaneish T Selvaraj, Siddharthan Chandran, Jared Sterneckert, Susanne Petri, Bettina Seeger, Michael Briese, Christian Stigloher, Thorsten Bischler, Andreas Hermann, Markus Damme, Michael Sendtner, Patrick Lüningschrör.

Increasing evidence suggests an essential function for autophagy in unconventional protein secretion (UPS). However, despite its relevance for the secretion of aggregate-prone proteins, the mechanisms of secretory autophagy in neurons have remained elusive. Here we show that the lower motoneuron disease-associated guanine exchange factor Plekhg5 drives the UPS of Sod1. Mechanistically, Sod1 is sequestered into autophagosomal carriers, which subsequently fuse with secretory lysosomal-related organelles (LROs). Exocytosis of LROs to release Sod1 into the extracellular milieu requires the activation of the small GTPase Rab26 by Plekhg5. Deletion of Plekhg5 in mice leads to the accumulation of Sod1 in LROs at swollen presynaptic sites. A reduced secretion of toxic ALS-linked SOD1G93A following deletion of Plekhg5 in SOD1G93A mice accelerated disease onset while prolonging survival due to an attenuated microglia activation. Using human iPSC-derived motoneurons we show that reduced levels of PLEKHG5 cause an impaired secretion of ALS-linked SOD1. Our findings highlight an unexpected pathophysiological mechanism that converges two motoneuron disease-associated proteins into a common pathway.

DOI: https://doi.org/10.1038/s41467-024-52875-5
Transl Psychiatry. 2024 Oct 01. 14(1): 397

Defective regulation of the eIF2-eIF2B translational axis underlies depressive-like behavior in mice and correlates with major depressive disorder in humans.

Alinny R Isaac, Mariana G Chauvet, Ricardo Lima-Filho, Beatriz de A Wagner, Bruno G Caroli, Renata E P Leite, Claudia K Suemoto, Paula Villela Nunes, Fernanda G De Felice, Sergio T Ferreira, Mychael V Lourenco.

Major depressive disorder (MDD) is a significant cause of disability in adults worldwide. However, the underlying causes and mechanisms of MDD are not fully understood, and many patients are refractory to available therapeutic options. Impaired control of brain mRNA translation underlies several neurodevelopmental and neurodegenerative conditions, including autism spectrum disorders and Alzheimer's disease (AD). Nonetheless, a potential role for mechanisms associated with impaired translational control in depressive-like behavior remains elusive. A key pathway controlling translation initiation relies on the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α-P) which, in turn, blocks the guanine exchange factor activity of eIF2B, thereby reducing global translation rates. Here we report that the expression of EIF2B5 (which codes for eIF2Bε, the catalytic subunit of eIF2B) is reduced in postmortem MDD prefrontal cortex from two distinct human cohorts and in the frontal cortex of social isolation-induced depressive-like behavior model mice. Further, pharmacological treatment with anisomycin or with salubrinal, an inhibitor of the eIF2α phosphatase GADD34, induces depressive-like behavior in adult C57BL/6J mice. Salubrinal-induced depressive-like behavior is blocked by ISRIB, a compound that directly activates eIF2B regardless of the phosphorylation status of eIF2α, suggesting that increased eIF2α-P promotes depressive-like states. Taken together, our results suggest that impaired eIF2-associated translational control may participate in the pathophysiology of MDD, and underscore eIF2-eIF2B translational axis as a potential target for the development of novel approaches for MDD and related mood disorders.

DOI: https://doi.org/10.1038/s41398-024-03128-y
Dis Model Mech. 2024 Sep 01. pii: dmm050635. [Epub ahead of print]17(9):

Activation of the heat shock response as a therapeutic strategy for tau toxicity.

Taylor R Stanley, Elizabeth M Otero, Amy L Knight, Aleen D Saxton, Xinxing Ding, Melissa Borgen, Brian C Kraemer, Karen S Kim Guisbert, Eric Guisbert.

  Alzheimer's disease is associated with the misfolding and aggregation of two distinct proteins, beta-amyloid and tau. Previously, it has been shown that activation of the cytoprotective heat shock response (HSR) pathway reduces beta-amyloid toxicity. Here, we show that activation of the HSR is also protective against tau toxicity in a cell-autonomous manner. Overexpression of HSF-1, the master regulator of the HSR, ameliorates the motility defect and increases the lifespan of transgenic C. elegans expressing human tau. By contrast, RNA interference of HSF-1 exacerbates the motility defect and shortens lifespan. Targeting regulators of the HSR also affects tau toxicity. Additionally, two small-molecule activators of the HSR, Geranylgeranylacetone (GGA) and Arimoclomol (AC), have substantial beneficial effects. Taken together, this research expands the therapeutic potential of HSR manipulation to tauopathies and reveals that the HSR can impact both beta-amyloid and tau proteotoxicity in Alzheimer's disease.

Keywords:   Caenorhabditis elegans ; Alzheimer’s disease; HSF1; Heat shock response; Tau (MAPT)

DOI:  https://doi.org/10.1242/dmm.050635
Mol Cell. 2024 Oct 03. pii: S1097-2765(24)00705-6. [Epub ahead of print]84(19): 3681-3691

Understanding nuclear mRNA export: Survival under stress.

Johanna Franziska Seidler, Katja Sträßer.

  Nuclear messenger RNA (mRNA) export is vital for cell survival under both physiological and stress conditions. To cope with stress, cells block bulk mRNA export while selectively exporting stress-specific mRNAs. Under physiological conditions, nuclear adaptor proteins recruit the mRNA exporter to the mRNA for export. By contrast, during stress conditions, the mRNA exporter is likely directly recruited to stress-specific mRNAs at their transcription sites to facilitate selective mRNA export. In this review, we summarize our current understanding of nuclear mRNA export. Importantly, we explore insights into the mechanisms that block bulk mRNA export and facilitate transcript-specific mRNA export under stress, highlighting the gaps that still need to be filled.

Keywords:  RBPs; RNA-binding proteins; bulk mRNA; export block; mRNA; nuclear mRNA export; stress conditions; stress-specific mRNA

DOI:  https://doi.org/10.1016/j.molcel.2024.08.028
PLoS Genet. 2024 Oct 03. 20(10): e1011424

The middle domain of Hsp104 can ensure substrates are functional after processing.

Hannah E Buchholz, Jane E Dorweiler, Sam Guereca, Brett T Wisniewski, James Shorter, Anita L Manogaran.

Molecular chaperones play a central role in protein disaggregation. However, the molecular determinants that regulate this process are poorly understood. Hsp104 is an AAA+ ATPase that disassembles stress granules and amyloids in yeast through collaboration with Hsp70 and Hsp40. In vitro studies show that Hsp104 processes different types of protein aggregates by partially translocating or threading polypeptides through the central pore of the hexamer. However, it is unclear how Hsp104 processing influences client protein function in vivo. The middle domain (MD) of Hsp104 regulates ATPase activity and interactions with Hsp70. Here, we tested how MD variants, Hsp104A503S and Hsp104A503V, process different protein aggregates. We establish that engineered MD variants fail to resolve stress granules but retain prion fragmentation activity required for prion propagation. Using the Sup35 prion protein, our in vitro and in vivo data indicate that the MD variants can disassemble Sup35 aggregates, but the disaggregated protein has reduced GTPase and translation termination activity. These results suggest that the middle domain can play a role in sensing certain substrates and plays an essential role in ensuring the processed protein is functional.

DOI: https://doi.org/10.1371/journal.pgen.1011424
Plant Physiol. 2024 Oct 01. pii: kiae513. [Epub ahead of print]

Functional Diversification of Sec13 Isoforms for Storage Protein Trafficking in Rice Endosperm Cells.

Yongfei Wang, Yulong Ren, Xuan Teng, Fan Wang, Yanyu Chen, Erchao Duan, Xin Wang, Tian Pan, Binglei Zhang, Gexing Wan, Yu Zhang, Pengcheng Zhang, Xiejun Sun, Wenkun Yang, Yun Zhu, Yu Chen, Wenjie Zhao, Xiaohang Han, Cailin Lei, Shanshan Zhu, Shijia Liu, Yihua Wang, Jianmin Wan.

  Coat protein complex II (COPII) vesicles play crucial roles in mediating the endoplasmic reticulum (ER) exit of newly synthesized proteins to the Golgi in eukaryotic cells. However, the molecular functions of COPII components and their functional diversifications in plant seeds remain obscure. Here, we showed that the rice (Oryza sativa) glutelin precursor accumulation12 (gpa12) mutant is defective in storage protein export from the ER, resulting in the formation of aggregated protein bodies. Map-based cloning revealed that GPA12 encodes a COPII outer layer protein, Sec13a, that mainly localizes to endoplasmic reticulum exit sites (ERES) and partially localizes to the Golgi. Biochemical experiments verified that Sec13a physically interacts with Sec31 and Sec16, and mutation in Sec13 compromises its interaction with Sec31 and Sec16, thereby affecting the membrane association of the inner complex components Sar1b and Sec23c. Apart from Sec13a, the rice genome encodes two other Sec13 isoforms, Sec13b and Sec13c. Notably, we observed an abnormal accumulation of globular ER structures in the sec13bc double mutant but not in the single mutants, suggesting a functional redundancy of Sec13b and Sec13c in modulating ER morphology. Taken together, our results substantiated that Sec13a plays an important role in regulating storage protein export from the ER, while Sec13b and Sec13c are required for maintaining ER morphology in rice endosperm cells. Our findings provide insights into the functional diversification of COPII components in plants.

Keywords:  COPII; Sec13 isoforms; functional diversification; rice; seed storage proteins

DOI:  https://doi.org/10.1093/plphys/kiae513
Sci Adv. 2024 Oct 04. 10(40): eadm9801

Single-molecule tracking reveals dynamic regulation of ribosomal scanning.

Hea Jin Hong, Antonia L Zhang, Adam B Conn, Gregor Blaha, Seán E O'Leary.

How eukaryotic ribosomes traverse messenger RNA (mRNA) leader sequences to search for protein-synthesis start sites remains one of the most mysterious aspects of translation and its regulation. While the search process is conventionally described by a linear "scanning" model, its exquisitely dynamic nature has restricted detailed mechanistic study. Here, we observed single Saccharomyces cerevisiae ribosomal scanning complexes in real time, finding that they scan diverse mRNA leaders at a rate of 10 to 20 nt s-1. We show that specific binding of a protein to its mRNA leader sequence substantially arrests scanning. Conversely, impairing scanning-complex guanosine 5'-triphosphate hydrolysis results in native start-site bypass. Our results illustrate an mRNA-centric, kinetically controlled regulatory model where the ribosomal pre-initiation complex amplifies a nuanced energetic landscape to regulate scanning and start-site selection fidelity.

DOI: https://doi.org/10.1126/sciadv.adm9801
J Cell Mol Med. 2024 Oct;28(19): e70125

KDELR2 is necessary for chronic obstructive pulmonary disease airway Mucin5AC hypersecretion via an IRE1α/XBP-1s-dependent mechanism.

Xiaojuan Wu, Fawang Du, Aijie Zhang, Guoyue Zhang, Rui Xu, Xianzhi Du.

  Airway mucus hypersecretion, a crucial pathological feature of chronic obstructive pulmonary disease (COPD), contributes to the initiation, progression, and exacerbation of this disease. As a macromolecular mucin, the secretory behaviour of Mucin5AC (MUC5AC) is highly dependent on a series of modifying and folding processes that occur in the endoplasmic reticulum (ER). In this study, we focused on the ER quality control protein KDEL receptor (KDELR) and demonstrated that KDELR2 and MUC5AC were colocalized in the airway epithelium of COPD patients and COPD model rats. In addition, knockdown of KDELR2 markedly reduced the expression of MUC5AC both in vivo and in vitro and knockdown of ATF6 further decreased the levels of KDELR2. Furthermore, pretreatment with 4μ8C, an IRE1α inhibitor, led to a partial reduction in the expression of KDELR2 and MUC5AC both in vivo and in vitro, which indicated the involvement of IRE1α/XBP-1s in the upstream signalling cascade. Our study revealed that KDELR2 plays a crucial role in airway MUC5AC hypersecretion in COPD, which might be dependent on ATF6 and IRE1α/XBP-1s upstream signalling.

Keywords:  COPD; KDELR2; Mucin5AC; UPR; endoplasmic reticulum

DOI:  https://doi.org/10.1111/jcmm.70125
Nat Commun. 2024 Sep 30. 15(1): 8448

A structural rationale for reversible vs irreversible amyloid fibril formation from a single protein.

Lukas Frey, Jiangtao Zhou, Gea Cereghetti, Marco E Weber, David Rhyner, Aditya Pokharna, Luca Wenchel, Harindranath Kadavath, Yiping Cao, Beat H Meier, Matthias Peter, Jason Greenwald, Roland Riek, Raffaele Mezzenga.

Reversible and irreversible amyloids are two diverging cases of protein (mis)folding associated with the cross-β motif in the protein folding and aggregation energy landscape. Yet, the molecular origins responsible for the formation of reversible vs irreversible amyloids have remained unknown. Here we provide evidence at the atomic level of distinct folding motifs for irreversible and reversible amyloids derived from a single protein sequence: human lysozyme. We compare the 2.8 Å structure of irreversible amyloid fibrils determined by cryo-electron microscopy helical reconstructions with molecular insights gained by solid-state NMR spectroscopy on reversible amyloids. We observe a canonical cross-β-sheet structure in irreversible amyloids, whereas in reversible amyloids, there is a less-ordered coexistence of β-sheet and helical secondary structures that originate from a partially unfolded lysozyme, thus carrying a "memory" of the original folded protein precursor. We also report the structure of hen egg-white lysozyme irreversible amyloids at 3.2 Å resolution, revealing another canonical amyloid fold, and reaffirming that irreversible amyloids undergo a complete conversion of the native protein into the cross-β structure. By combining atomic force microscopy, cryo-electron microscopy and solid-state NMR, we show that a full unfolding of the native protein precursor is a requirement for establishing irreversible amyloid fibrils.

DOI: https://doi.org/10.1038/s41467-024-52681-z
J Am Chem Soc. 2024 Oct 03.

Chemical Diversification of Enzymatically Assembled Polyubiquitin Chains to Decipher the Ubiquitin Codes Programmed on the Branch Structure.

Takafumi Furuhata, Bumkyu Choi, Taiki Uno, Ryota Shinohara, Yusuke Sato, Kei Okatsu, Shuya Fukai, Akimitsu Okamoto.

The multimerization of ubiquitins at different positions of lysine residues to form heterotypic polyubiquitin chains is a post-translational modification that is essential for the precise regulation of protein functions and degradative fates in living cells. The understanding of structure-activity relationships underlying their diverse properties has been seriously impeded by difficulties in the preparation of a series of folded heterotypic chains appropriately functionalized with different chemical tags for the systematic evaluation of their multifaceted functions. Here, we report a chemical diversification of enzymatically assembled polyubiquitin chains that enables the facile preparation of folded heterotypic chains with different functionalities. By introducing an acyl hydrazide at the C terminus of the proximal ubiquitin, polyubiquitin chains were readily diversified from the same starting materials with a variety of molecules, ranging from small molecules to biopolymers, under nondenaturing conditions. This chemical diversification allowed the systematic study of the functional differences of K63/K48 heterotypic chains based on the position of the branch point during enzymatic deubiquitination and proteasomal proteolysis, thus demonstrating critical roles of the branch position in both the positive and negative control of ubiquitin-mediated reactions. The chemical diversification of the heterotypic chains provides a robust chemical platform to reframe the understanding of how the ubiquitin codes are regulated from the viewpoint of the branch structure for the precise control of cell functions, which has not been deciphered solely on the basis of the linkage types.

DOI: https://doi.org/10.1021/jacs.4c11279
bioRxiv. 2024 Sep 16. pii: 2024.09.13.612970. [Epub ahead of print]

Translational profiling of stress-induced small proteins uncovers an unexpected connection among distinct signaling systems.

Sangeevan Vellappan, Junhong Sun, John Favate, Pranavi Jagadeesan, Debbie Cerda, Premal Shah, Srujana S Yadavalli.

Signaling networks in bacteria enable sensing and adaptation to challenging environments by activating specific genes that help counteract stressors. Small proteins (≤ 50 amino acids long) are a rising class of bacterial stress response regulators. Escherichia coli encodes over 150 small proteins, most of which lack known phenotypes and their biological roles remain elusive. Using magnesium limitation as a stressor, we investigate small proteins induced in response to stress using ribosome profiling, RNA sequencing, and transcriptional reporter assays. We uncover 17 small proteins with increased translation initiation, a majority of which are transcriptionally upregulated by the PhoQ-PhoP two-component signaling system, crucial for magnesium homeostasis. Next, we describe small protein-specific deletion and overexpression phenotypes, which underscore the physiological significance of their expression in low magnesium stress. Most remarkably, our study reveals that a small membrane protein YoaI is an unusual connector of the major signaling networks - PhoR-PhoB and EnvZ-OmpR in E. coli , advancing our understanding of small protein regulators of cellular signaling.
Highlights: Ribo-RET identifies 17 small proteins induced under low Mg 2+ stress in E. coli Many of these proteins are transcriptionally activated by PhoQP signaling systemHalf of the stress-induced small proteins localize to the membraneDeletion or overexpression of specific small proteins affects growth under stressSmall protein YoaI connects PhoR-PhoB and EnvZ-OmpR signaling networks.
Graphical abstract:

DOI: https://doi.org/10.1101/2024.09.13.612970
EMBO Rep. 2024 Oct 02.

Ageing-associated long non-coding RNA extends lifespan and reduces translation in non-dividing cells.

Shajahan Anver, Ahmed Faisal Sumit, Xi-Ming Sun, Abubakar Hatimy, Konstantinos Thalassinos, Samuel Marguerat, Nazif Alic, Jürg Bähler.

  Genomes produce widespread long non-coding RNAs (lncRNAs) of largely unknown functions. We characterize aal1 (ageing-associated lncRNA), which is induced in quiescent fission yeast cells. Deletion of aal1 shortens the chronological lifespan of non-dividing cells, while ectopic overexpression prolongs their lifespan, indicating that aal1 acts in trans. Overexpression of aal1 represses ribosomal-protein gene expression and inhibits cell growth, and aal1 genetically interacts with coding genes functioning in protein translation. The aal1 lncRNA localizes to the cytoplasm and associates with ribosomes. Notably, aal1 overexpression decreases the cellular ribosome content and inhibits protein translation. The aal1 lncRNA binds to the rpl1901 mRNA, encoding a ribosomal protein. The rpl1901 levels are reduced ~2-fold by aal1, which is sufficient to extend lifespan. Remarkably, the expression of the aal1 lncRNA in Drosophila boosts fly lifespan. We propose that aal1 reduces the ribosome content by decreasing Rpl1901 levels, thus attenuating the translational capacity and promoting longevity. Although aal1 is not conserved, its effect in flies suggests that animals feature related mechanisms that modulate ageing, based on the conserved translational machinery.

Keywords:   Schizosaccharomyces pombe ; Chronological Lifespan; Protein Translation; RNA Regulation; Ribosomal Protein

DOI:  https://doi.org/10.1038/s44319-024-00265-9
STAR Protoc. 2024 Sep 28. pii: S2666-1667(24)00513-6. [Epub ahead of print]5(4): 103348

Protocol to measure and analyze protein interactions in mammalian cells using bioluminescence resonance energy transfer.

Carla Jane Duval, Candy Laura Steffen, Karolina Pavic, Daniel Kwaku Abankwa.

  Bioluminescence resonance energy transfer (BRET) allows to quantitate protein interactions in intact cells. Here, we present a protocol for measuring BRET due to transient interactions of oncogenic K-RasG12V in plasma membrane nanoclusters of HEK293-EBNA cells. We describe steps for seeding, transfecting, and replating cells. We then detail procedures for their preparation for BRET measurements on a CLARIOstar microplate reader and detailed data analysis. For complete details on the use and execution of this protocol, please refer to Steffen et al.1.

Keywords:  Cell Biology; Cell-based Assays; Molecular Biology

DOI:  https://doi.org/10.1016/j.xpro.2024.103348
Cell Rep. 2024 Oct 03. pii: S2211-1247(24)01135-5. [Epub ahead of print]43(10): 114784

Proteome-wide CETSA reveals diverse apoptosis-inducing mechanisms converging on an initial apoptosis effector stage at the nuclear periphery.

Anderson Daniel Ramos, Ying Yu Liang, Olga Surova, Smaranda Bacanu, Marc-Antoine Gerault, Tamoghna Mandal, Sophia Ceder, Anette Langebäck, Albin Österroos, George A Ward, Jonas Bergh, Klas G Wiman, Sören Lehmann, Nayana Prabhu, Sara Lööf, Pär Nordlund.

  Cellular phenotypes of apoptosis, as well as the activation of apoptosis caspase cascades, are well described. However, sequences and locations of early biochemical effector events after apoptosis initiation are still only partly understood. Here, we use integrated modulation of protein interaction states-cellular thermal shift assay (IMPRINTS-CETSA) to dissect the cellular biochemistry of early stages of apoptosis at the systems level. Using 5 families of cancer drugs and a new CETSA-based method to monitor the cleavage of caspase targets, we discover the initial biochemistry of the effector stage of apoptosis for all the studied drugs being focused on the peripheral nuclear region rather than the cytosol. Despite very different candidate apoptosis-inducing mechanisms of the drug families, as revealed by the CETSA data, they converge into related biochemical modulations in the peripheral nuclear region. This implies a higher control of the localization of the caspase cascades than previously anticipated and highlights the nuclear periphery as a critical vulnerability for cancer therapies.

Keywords:  CETSA; CP: Cancer; CP: Cell biology; IAP inhibitors; PI3K inhibitors; apoptosis; cancer drugs; caspase cleavage; glutathione metabolism; mass spectrometry; proteomics; taxanes

DOI:  https://doi.org/10.1016/j.celrep.2024.114784
bioRxiv. 2024 Sep 22. pii: 2024.09.18.613588. [Epub ahead of print]

Family-Wide Photoproximity Profiling of Integrin Protein Social Networks in Cancer.

Anthony J Carlos, Dongbo Yang, Deborah M Thomas, Shuyuan Huang, Keira I Harter, Raymond E Moellering.

Integrin family transmembrane receptors mediate dynamic interactions between cells and their extracellular microenvironment. The heterogeneous interaction partners of integrins directly regulate cell adhesion, motility, proliferation, and intracellular signaling. Despite the recognized importance of protein-protein interactions and the formation of signaling hubs around integrins, the ability to detect and quantify these dynamic binding partners with high spatial and temporal resolution remains challenging. Here, we developed an integrin-family-directed quantitative photoproximity protein interaction (PhotoPPI) profiling method to detect and quantify native integrin-centered protein social networks on live cells and tissues without the need for genetic manipulation, antibodies, or non-physiologic cell culture conditions. We drafted quantitative maps of integrin-centered protein social networks, highlighting conserved and unique binding partners between different cell types and cellular microenvironments. Comparison of integrin social networks in cancer cell lines of diverse tissue of origin and disease state identified specific AND-gate binding partners involved cell migration, microenvironmental interactions and proliferation that serve as markers of tumor cell metastatic state. Finally, we identified unique combinations - or barcodes - of integrin-proximal proteins on the surface of pre- and post-metastatic triple negative breast cancer (TNBC) cells whose expression strongly correlate with both positive and negative disease progression and outcomes in TNBC patients. Taken together, these data provide the first family-wide high-resolution maps of native protein interactors on live cells and identify dynamic integrin-centered social networks as potential AND-gate markers of cell identity, microenvironmental context and disease state.

DOI: https://doi.org/10.1101/2024.09.18.613588
Mol Cell. 2024 Oct 03. pii: S1097-2765(24)00738-X. [Epub ahead of print]84(19): 3692-3705

RNA-driven phase transitions in biomolecular condensates.

Gable M Wadsworth, Sukanya Srinivasan, Lien B Lai, Moulisubhro Datta, Venkat Gopalan, Priya R Banerjee.

  RNAs and RNA-binding proteins can undergo spontaneous or active condensation into phase-separated liquid-like droplets. These condensates are cellular hubs for various physiological processes, and their dysregulation leads to diseases. Although RNAs are core components of many cellular condensates, the underlying molecular determinants for the formation, regulation, and function of ribonucleoprotein condensates have largely been studied from a protein-centric perspective. Here, we highlight recent developments in ribonucleoprotein condensate biology with a particular emphasis on RNA-driven phase transitions. We also present emerging future directions that might shed light on the role of RNA condensates in spatiotemporal regulation of cellular processes and inspire bioengineering of RNA-based therapeutics.

Keywords:  RNA chaperones; RNP granules; gene regulation; helicases; liquid-liquid phase separation; percolation; stress granules

DOI:  https://doi.org/10.1016/j.molcel.2024.09.005
Nat Commun. 2024 Oct 04. 15(1): 8621

Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors.

Jan Silhan, Pavla Fajtova, Jitka Bartosova, Brianna M Hurysz, Jehad Almaliti, Yukiko Miyamoto, Lars Eckmann, William H Gerwick, Anthony J O'Donoghue, Evzen Boura.

The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB's low specificity for Tv20S compared to the human proteasome and demonstrate CP-17's higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis.

DOI: https://doi.org/10.1038/s41467-024-53022-w
bioRxiv. 2024 Sep 17. pii: 2024.09.16.613361. [Epub ahead of print]

High-throughput optimized prime editing mediated endogenous protein tagging for pooled imaging of protein localization.

Henry M Sanchez, Tomer Lapidot, Ophir Shalem.

The subcellular organization of proteins carries important information on cellular state and gene function, yet currently there are no technologies that enable accurate measurement of subcellular protein localizations at scale. Here we develop an approach for pooled endogenous protein tagging using prime editing, which coupled with an optical readout and sequencing, provides a snapshot of proteome organization in a manner akin to perturbation-based CRISPR screens. We constructed a pooled library of 17,280 pegRNAs designed to exhaustively tag 60 endogenous proteins spanning diverse localization patterns and explore a large space of genomic and pegRNA design parameters. Pooled measurements of tagging efficiency uncovered both genomic and pegRNA features associated with increased efficiency, including epigenetic states and interactions with transcription. We integrate pegRNA features into a computational model with predictive value for tagging efficiency to constrain the design space of pegRNAs for large-scale peptide knock-in. Lastly, we show that combining in-situ pegRNA sequencing with high-throughput deep learning image analysis, enables exploration of subcellular protein localization patterns for many proteins in parallel following a single pooled lentiviral transduction, setting the stage for scalable studies of proteome dynamics across cell types and environmental perturbations.

DOI: https://doi.org/10.1101/2024.09.16.613361